Field of the Invention
The present invention relates to the fields of molecular biology, virology, protein synthesis, gene therapy, and medicine. The invention provides novel mRNA regulatory elements and methods for controlling protein expression in selected mammalian host cells. In particular embodiments, the invention provides compositions including polyA-deleted rAAV vectors that express a stably-produced mRNA that includes an inverted terminal repeat sequence at its 3-end, which forms an element having the ability to form a hairpin, double-stranded RNA structure that is independent of other non-coding RNAs such as micoRNA, siRNA, etc. mRNAs containing the stable hairpin structure lack polyadenylation signals, which allow minimal protein translation. In the presence of specific proteins, however, high levels of protein translation can be obtained, and precisely controlled. The resulting vectors are particularly useful in diagnostic and/or therapeutic regimens, including, for example, the treatment of one or more mammalian disorders or diseases, and in particular, for treating defects resulting from a decrease in, or an absence of expression of one or more particular polypeptides. Also provided are methods for preparing rAAV vector-based medicaments for use in viral vector-based gene therapies.
Description of Related Art
Two major mechanisms or strategies exist for controlling protein expression in mammalian cells. One is the use of tissue-specific promoters; the other is the use of siRNA. However, these methods rely upon regulation at the transcriptional level (i.e., the process of synthesizing mRNA from its DNA template), and not upon regulation at the translational level (i.e., synthesizing the encoded protein from the mRNA message). Conventional siRNA-based methods have focused on the inhibition of translation of mRNA into protein (i.e., decreasing the amount of protein produced).
What is lacking in the prior art, however, are methods that permit the enhancement, i.e., an increase, of protein translation from mRNAs.